Cloning and Characterization of Methenyltetrahydrofolate Synthetase from Saccharomyces cerevisiae

Abstract

The folate derivative 5-formyltetrahydrofolate (folinic acid; 5-CHO-THF) was discovered over 40 years ago, but its role in metabolism remains poorly understood. Only one enzyme is known that utilizes 5-CHO-THF as a substrate: 5,10-methenyltetrahydrofolate synthetase (MTHFS). A BLAST search of the yeast genome using the human MTHFS sequence revealed a 211-amino acid open reading frame (YER183c) with significant homology. The yeast enzyme was expressed in Escherichia coli, and the purified recombinant enzyme exhibited kinetics similar to previously purified MTHFS. No new phenotype was observed in strains disrupted at MTHFS or in strains additionally disrupted at the genes encoding one or both serine hydroxymethyltransferases (SHMT) or at the genes encoding one or both methylenetetrahydrofolate reductases. However, when the MTHFS gene was disrupted in a strain lacking the de novo folate biosynthesis pathway, folinic acid (5-CHO-THF) could no longer support the folate requirement. We have thus named the yeast gene encoding methenyltetrahydrofolate synthetase FAU1 (folinic acid utilization). Disruption of the FAU1 gene in a strain lacking both 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase isozymes (ADE16 and ADE17) resulted in a growth deficiency that was alleviated by methionine. Genetic analysis suggested that intracellular accumulation of the purine intermediate AICAR interferes with a step in methionine biosynthesis. Intracellular levels of 5-CHO-THF were determined in yeast disrupted at FAU1 and other genes encoding folate-dependent enzymes. In fau1 disruptants, 5-CHO-THF was elevated 4-fold over wild-type yeast. In yeast lacking MTHFS along with both AICAR transformylases, 5-CHO-THF was elevated 12-fold over wild type. 5-CHO-THF was undetectable in strains lacking SHMT activity, confirming SHMT as the in vivo source of 5-CHO-THF. Taken together, these results indicate that S. cerevisiae harbors a single, nonessential, MTHFS activity. Growth phenotypes of multiply disrupted strains are consistent with a regulatory role for 5-CHO-THF in one-carbon metabolism and additionally suggest a metabolic interaction between the purine and methionine pathways.Although 5-formyl-tetrahydrofolate (5-CHO-THF 1 ; folinic acid or leucovorin) is the most stable derivative of the reduced folates (1), it does not have a known direct role as a one-carbon donor. Leucovorin has been employed extensively as a rescue agent in chemotherapeutic protocols, but the physiological role of this folate derivative remains poorly understood. 5-CHO-THF can arise nonenzymatically from the hydrolysis of 5,10-methenyl-THF (5,10-CH ϩ -THF) under mild conditions (2). For this reason, it was considered likely that the 5-CHO-THF detected in cell extracts was an artifact of preparation rather than a normal component of folate metabolism. More recent studies designed to prevent the possibility of artifactual conversion concluded that 5-CHO-THF is indeed a naturally occurring metabolite (3). The biological source of 5-CHO-THF, however,...

Cloning and Characterization of Mitochondrial 5-Formyltetrahydrofolate Cycloligase from Higher Plants

“…5-CHO-H 4 PteGlu n normally represents only 3-10% of the total cellular folate pool in mammals and yeast (4,7), whereas values of 14 -40% are reported for leaves and other metabolically active plant tissues (16,17). Moreover, 5-CHO-H 4 PteGlu n makes up 50% of the mitochondrial folate pool in pea leaves (17), which is much more than in mammalian mitochondria (18 -20).…”

“…Its likely main source is hydrolysis of 5,10-CHϭH 4 PteGlu n catalyzed by a side reaction of serine hydroxymethyltransferase (SHMT) in the presence of glycine (4,12), although some spontaneous chemical hydrolysis of 5,10-CHϭH 4 PteGlu n in mildly acidic subcellular compartments is also possible (13). Unlike other folate species, 5-CHO-H 4 PteGlu n does not serve as a one-carbon (C 1 ) donor, but is a potent inhibitor of SHMT and several other enzymes of C 1 metabolism (1). It is therefore considered that 5-CHO-H 4 PteGlu n is a potential regulator of C 1 metabolism, and that its cellular concentration is set by a futile cycle comprising its synthesis by SHMT and its reconversion to 5,10-CHϭH 4 PteGlu n by 5-FCL.…”

“…Unlike other folate species, 5-CHO-H 4 PteGlu n does not serve as a one-carbon (C 1 ) donor, but is a potent inhibitor of SHMT and several other enzymes of C 1 metabolism (1). It is therefore considered that 5-CHO-H 4 PteGlu n is a potential regulator of C 1 metabolism, and that its cellular concentration is set by a futile cycle comprising its synthesis by SHMT and its reconversion to 5,10-CHϭH 4 PteGlu n by 5-FCL. Specific roles assigned to this cycle in mammalian cells include regulation of de novo purine synthesis (14), of the flux of C 1 units from serine into the folate pool (15), and of homocysteine remethylation (7).…”

“…5-FCL has been purified from bacteria and mammalian liver (1) and cloned from rabbit (2), human (3), and yeast (4). The native proteins from these species are all monomers of 23-28 kDa (4 -6), and the bacterial and mammalian enzymes have been shown to be specific for the 6S form of 5-CHO-H 4 PteGlu n (5,6). Mammalian 5-FCLs appear to be cytosolic enzymes (2,7), although a small amount of activity (15% of total) was reported in mitochondria from human liver (8).…”

Homocysteine accumulation causes a defect in purine biosynthesis: further characterization of Schizosaccharomyces pombe methionine auxotrophs

“…5a), suggesting a metabolic interaction between the methionine and purine biosynthetic pathways. Holmes & Appling (2002) reported that intracellular accumulation of the purine intermediate 59-phosphoribosyl-5-aminoimidazole-4-carboxamide (AICAR) interferes with methionine biosynthesis in Sac. cerevisiae.…”